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1.  Bladder Cancer Biomarker Discovery Using Global Metabolomic Profiling of Urine 
PLoS ONE  2014;9(12):e115870.
Bladder cancer (BCa) is a common malignancy worldwide and has a high probability of recurrence after initial diagnosis and treatment. As a result, recurrent surveillance, primarily involving repeated cystoscopies, is a critical component of post diagnosis patient management. Since cystoscopy is invasive, expensive and a possible deterrent to patient compliance with regular follow-up screening, new non-invasive technologies to aid in the detection of recurrent and/or primary bladder cancer are strongly needed. In this study, mass spectrometry based metabolomics was employed to identify biochemical signatures in human urine that differentiate bladder cancer from non-cancer controls. Over 1000 distinct compounds were measured including 587 named compounds of known chemical identity. Initial biomarker identification was conducted using a 332 subject sample set of retrospective urine samples (cohort 1), which included 66 BCa positive samples. A set of 25 candidate biomarkers was selected based on statistical significance, fold difference and metabolic pathway coverage. The 25 candidate biomarkers were tested against an independent urine sample set (cohort 2) using random forest analysis, with palmitoyl sphingomyelin, lactate, adenosine and succinate providing the strongest predictive power for differentiating cohort 2 cancer from non-cancer urines. Cohort 2 metabolite profiling revealed additional metabolites, including arachidonate, that were higher in cohort 2 cancer vs. non-cancer controls, but were below quantitation limits in the cohort 1 profiling. Metabolites related to lipid metabolism may be especially interesting biomarkers. The results suggest that urine metabolites may provide a much needed non-invasive adjunct diagnostic to cystoscopy for detection of bladder cancer and recurrent disease management.
PMCID: PMC4277370  PMID: 25541698
The Journal of urology  2011;186(1):66-72.
Introduction and Objective
Increased body mass index (BMI) is associated with worse outcomes for several different malignancies. The relationship between BMI and urothelial carcinoma is poorly understood. We investigated the association between BMI and oncological outcomes in upper tract urothelial carcinoma (UTUC).
We retrospectively studied 520 patients treated with radical nephroureterectomy (RNU) for UTUC without neoadjuvant chemotherapy. Univariable Cox regression analyses were performed to evaluate recurrence-free (RFS) and cancer-specific survival (CSS) estimates. We created a multivariable model based on preoperative and postoperative characteristics. BMI was treated as a continuous and a categorical variable defined as normal weight (<25 kg/m2), overweight (25–29.9 kg/m2) or obese (≥30 kg/m2).
The median patient BMI was 27.9 kg/m2 (IQR: 6.7). Median follow-up was 38 months (IQR: 54). Patients with a higher BMI were more likely to have infiltrative architecture (p<0.001) and lymphovascular invasion (p=0.012). BMI was not associated with age, smoking history, pathologic stage, tumor grade, concomitant CIS, tumor necrosis, and the selection of a laparoscopic or open procedure. In the preoperative multivariable model a BMI of 25–29 kg/m2 (HR 2.25, 95% CI: 1.3–3.8, p=0.003) and BMI ≥ 30 kg/m2 (HR 3.72, 95% CI: 2.2–6.3, <0.001) were both associated with disease recurrence. A BMI ≥30kg/m2 (HR 4.24, CI: 2.4–7.5, p<0.001) was associated with cancer specific death. In the postoperative model, tumor stage (p<0.001), positive lymph nodes (HR 2.52, 95% CI: 1.59–4.0, p<0.001), BMI 25–29 kg/m2 (HR 2.18, 95% CI: 1.27–3.73, p=0.005) and BMI ≥30 kg/m2 (HR 3.52, 95% CI: 2.08–5.95, p<0.001) were associated with disease recurrence. Tumor stage (p<0.001), positive lymph nodes (HR 3.1, 95% CI: 1.84–5.21, p<0.001), and a BMI≥30 kg/m2 (HR 4.13, 95% CI: 2.32–7.36, p<0.001) were associated with cancer-specific death.
Higher BMI is associated with worse cancer-specific outcomes in patients treated with RNU for UTUC. Improving cancer specific survival by also focusing on patient modifiable factors such as BMI could have significant individual and public health implications in UTUC patients. Future studies should be encouraged to evaluate the molecular mechanisms of tumorigenesis and progression associated with metabolic changes in obesity.
PMCID: PMC4170670  PMID: 21571333
Obesity; BMI; Upper Urinary Tract; Urothelial; Carcinoma
3.  Triglycerides in the Human Kidney Cortex: Relationship with Body Size 
PLoS ONE  2014;9(8):e101285.
Obesity is associated with increased risk for kidney disease and uric acid nephrolithiasis, but the pathophysiological mechanisms underpinning these associations are incompletely understood. Animal experiments have suggested that renal lipid accumulation and lipotoxicity may play a role, but whether lipid accumulation occurs in humans with increasing body mass index (BMI) is unknown. The association between obesity and abnormal triglyceride accumulation in non-adipose tissues (steatosis) has been described in the liver, heart, skeletal muscle and pancreas, but not in the human kidney. We used a quantitative biochemical assay to quantify triglyceride in normal kidney cortex samples from 54 patients undergoing nephrectomy for localized renal cell carcinoma. In subsets of the study population we evaluated the localization of lipid droplets by Oil Red O staining and measured 16 common ceramide species by mass spectrometry. There was a positive correlation between kidney cortex trigyceride content and BMI (Spearman R = 0.27, P = 0.04). Lipid droplets detectable by optical microscopy had a sporadic distribution but were generally more prevalent in individuals with higher BMI, with predominant localization in proximal tubule cells and to a lesser extent in glomeruli. Total ceramide content was inversely correlated with triglycerides. We postulate that obesity is associated with abnormal triglyceride accumulation (steatosis) in the human kidney. In turn, steatosis and lipotoxicity may contribute to the pathogenesis of obesity-associated kidney disease and nephrolithiasis.
PMCID: PMC4149342  PMID: 25170827
4.  A gain of function mutation in DHT synthesis in castration-resistant prostate cancer 
Cell  2013;154(5):1074-1084.
Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation leading to a gain-of-function in 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), which catalyzes the initial rate-limiting step in the conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3βHSD1 is a valid target for the treatment of CRPC.
PMCID: PMC3931012  PMID: 23993097
5.  Impact of Smoking on Oncologic Outcomes of Upper Tract Urothelial Carcinoma After Radical Nephroureterectomy 
European urology  2012;63(6):1082-1090.
Ci garette smoking is a common risk factor for developing upper tract urothelial carcinoma (UTUC).
To assess the impact of cigarette smoking status, cumulative smoking exposure, and time from cessation on oncologic UTUC outcomes in patients treated with radical nephroureterectomy (RNU).
Design, setting, and participants
A total of 864 patients underwent RNU at five institutions. The median follow-up in this retrospective study was 50 mo. Smoking history included smoking status, quantity of cigarettes per day (CPD), duration in years, and years from smoking cessation. The cumulative smoking exposure was categorized as light-short-term (≤19 CPD and ≤19.9 yr), moderate (all combinations except light-short-term and heavy-long-term), and heavy-long-term (≥ 20CPD and ≥ 20 yr).
RNU with or without lymph node dissection. No patient received neoadjuvant chemotherapy.
Outcome measurements and statistical analysis
Univariable and multivariable logistic regression and competing risk regression analyses assessed the effects of smoking on oncologic outcomes.
Results and limitations
A total of 244 patients (28.2%) never smoked; 297 (34.4%) and 323 (37.4%) were former and current smokers, respectively. Among smokers, 87 (10.1%), 331 (38.3%), and 202 (23.4%) were light-short-term, moderate, and heavy-long-term smokers, respectively. Current smoking status, smoking ≥20CPD ≥20 yr, and heavy-long-term smoking were associated with advanced disease (p values ≤0.004), greater likelihood of disease recurrence (p values ≤0.01), and cancer-specific mortality (p values ≤0.05) on multivariable analyses that adjusted for standard features. Patients who quit smoking ≥10 yr prior to RNU did not differ from never smokers regarding advanced tumor stages, disease recurrence, and cancer-specific mortality, but they had better oncologic outcomes then current smokers and those patients who quit smoking <10 yr prior to RNU. The study is limited by its retrospective nature.
Cigarette smoking is significantly associated with advanced disease stages, disease recurrence, and cancer-specific mortality in patients treated with RNU for UTUC. Current smokers and those with a heavy and long-term smoking exposure have the highest risk for poor oncologic outcomes. Smoking cessation >10 yr prior to RNU seems to mitigate some detrimental effects. These results underscore the need for smoking cessation and prevention programs.
PMCID: PMC3969987  PMID: 22743166
Smoking; Urothelial carcinoma; Transitional cell carcinoma; Upper urinary tract; Radical nephroureterectomy; Dose–response relationship; Recurrence; Survival; Prognosis
6.  Impact of Smoking and Smoking Cessation on Oncologic Outcomes in Primary Non–muscle-invasive Bladder Cancer 
European urology  2012;63(4):724-732.
Cigarette smoking is the best-established risk factor for urothelial carcinoma (UC) development, but the impact on oncologic outcomes remains poorly understood.
To analyse the effects of smoking status, cumulative exposure, and time from smoking cessation on the prognosis of patients with primary non–muscle-invasive bladder cancer (NMIBC).
Design, setting, and participants
We collected smoking data from 2043 patients with primary NMIBC. Smoking variables included smoking status, average number of cigarettes smoked per day (CPD), duration in years, and time since smoking cessation. Lifetime cumulative smoking exposure was categorised as light short term (≤19 CPD, ≤19.9 yr), light long term (≤19 CPD, ≥20 yr), heavy short term (≥20 CPD, ≤19.9 yr) and heavy long term (≥20 CPD, ≥20 yr). The median follow-up in this retrospective study was 49 mo.
Transurethral resection of the bladder with or without intravesical instillation therapy.
Outcome measurements and statistical analysis
Univariable and multivariable logistic regression and competing risk regression analyses assessed the effects of smoking on outcomes.
Results and limitations
There was no difference in clinicopathologic factors among never (24%), former (47%), and current smokers (29%). Smoking status was associated with the cumulative incidence of disease progression in multivariable analysis (p = 0.003); current smokers had the highest cumulative incidences. Among current and former smokers, cumulative smoking exposure was associated with disease recurrence (p < 0.001), progression (p < 0.001), and overall survival (p < 0.001) in multivariable analyses that adjusted for the effects of standard clinicopathologic factors and smoking status; heavy long-term smokers had the worst outcomes, followed by light long-term, heavy short-term, and light short-term smokers. Smoking cessation >10 yr reduced the risk of disease recurrence (hazard ratio [HR]: 0.66; 95% confidence interval [CI], 0.52–0.84; p < 0.001) and progression (HR: 0.42; 95% CI, 0.22–0.83; p = 0.036) in multivariable analyses. The study is limited by its retrospective nature.
Smoking status and a higher cumulative smoking exposure are associated with worse prognosis in patients with NMIBC. Smoking cessation >10 yr abrogates this detrimental effect. These findings underscore the need for integrated smoking cessation and prevention programmes in the management of NMIBC patients.
PMCID: PMC3969986  PMID: 22925575
Smoking; Urothelial carcinoma; Non–muscle-invasive bladder cancer; Recurrence; Progression; Survival; Dose–response relationship
7.  BAP1 loss defines a new class of renal cell carcinoma 
Nature genetics  2012;44(7):751-759.
The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes including BAP1. BAP1, a nuclear deubiquitinase, is inactivated in 15% of clear-cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation, but not H2AK119ub1 deubiquitination. BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Interestingly, BAP1 and PBRM1 mutations anticorrelate in tumors (P=3×10−5), and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q=0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q=0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.
PMCID: PMC3788680  PMID: 22683710
8.  Risk of cancer-specific mortality following recurrence after radical nephroureterectomy 
Annals of surgical oncology  2012;19(13):4337-4344.
To describe the natural history and identify predictors of cancer-specific survival in patients who experience disease recurrence after radical nephroureterectomy (RNU) for upper tract urothelial carcinoma (UTUC).
Of 2,494 UTUC patients treated with RNU without neoadjuvant chemotherapy, 597 patients experienced disease recurrence. 148 patients (25%) received adjuvant chemotherapy before disease recurrence. Multivariable Cox regression model addressed time to cancer-specific mortality after disease recurrence.
The median time from RNU to disease recurrence was 12 months (IQR 5–22). 491 of 597 (82%) patients died from UTUC and 8 patients (1.3%) died from other causes. The median time from disease recurrence to death of UTUC was 10 months. Actuarial cancer-specific survival estimate at 12 months after disease recurrence was 35%. On multivariable analysis that adjusted for the effects of standard clinico-pathologic characteristics, higher tumor stages (HR pT3 vs. pT0-T1: 1.66, p=0.001; HR pT4 vs. pT0-T1: 1.90, p=0.002), absence of lymph node dissection (HR 1.28, p=0.041), ureteral tumor location (HR 1.44, p<0.0005) and a shorter interval from surgery to disease recurrence (p<0.0005) were significantly associated with cancer-specific mortality. The adjusted 6, 12 and 24 months post-recurrence cancer-specific mortality was 73%, 60% and 57%, respectively.
Approximately 80% of patients who experience disease recurrence after RNU die within two years post-recurrence. Patients with non-organ-confined stage, absence of lymph node dissection, ureteral tumor location and/or shorter time to disease recurrence died of their tumor faster than their counterparts. These factors should be considered in patient counseling and risk-stratification for salvage treatment decision-making.
PMCID: PMC3576920  PMID: 22805867
urothelial carcinoma; upper urinary tract; recurrence; survival; prognosis
9.  A Validated Tumorgraft Model Reveals Activity of Dovitinib Against Renal Cell Carcinoma 
Science translational medicine  2012;4(137):137ra75.
Most anticancer drugs entering clinical trials fail to achieve approval from the US FDA. Drug development is hampered by the lack of preclinical models with therapeutic predictive value. Herein, we report the development and validation of a tumorgraft model of renal cell carcinoma (RCC) and its application to the evaluation of an experimental drug. Tumor samples from 94 patients were implanted in the kidney of mice without additives or disaggregation. Tumors from 35 patients formed tumorgrafts, and 16 stable lines were established. Samples from metastatic sites engrafted at high frequency, and stable engraftment of primary tumors in mice correlated with decreased patient survival suggesting that tumor growth in mice may reveal the acquisition by the tumor of an ability to thrive at distant sites and metastasize. Tumorgrafts retained the histology, gene expression, DNA copy number alterations, and over 90% of the protein-coding gene mutations of the corresponding tumors. As determined by the induction of hypercalcemia in tumorgraft-bearing mice, tumorgrafts were able to act on the host causing paraneoplastic syndromes. In studies simulating drug exposures in patients, RCC tumorgraft growth was inhibited by sunitinib and sirolimus (into which temsirolimus is converted in humans), but not by erlotinib, which was used as a control. Dovitinib, a drug in clinical development, showed greater activity than sunitinib and sirolimus. The routine incorporation of models recapitulating the molecular genetics and drug sensitivities of human tumors into preclinical programs has the potential to improve oncology drug development.
PMCID: PMC3570965  PMID: 22674553
xenograft; tumor graft; orthotopic; PK; NOD/SCID; kidney cancer; clear-cell renal cell carcinoma
10.  Metabolomic Profiling Reveals Potential Markers and Bioprocesses Altered in Bladder Cancer Progression 
Cancer research  2011;71(24):7376-7386.
While alterations in xenobiotic metabolism are considered causal in the development of bladder cancer (BCa), the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in BCa. This metabolic signature distinguished both normal and benign bladder from BCa. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing BCa from controls, and also non-muscle from muscle-invasive BCa. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in BCa. In particular, we validated tumor-associated hypermethylation in the CYP1A1 and CYP1B1 promoters of BCa tissues by bisulfite sequence analysis and methylation-specific PCR, and also by in vitro treatment of T-24 BCa cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Further, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of BCa specimens compared to matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of BCa, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.
PMCID: PMC3249241  PMID: 21990318
11.  Interplay between pVHL and mTORC1 pathways in clear-cell renal cell carcinoma 
Molecular cancer research : MCR  2011;9(9):1255-1265.
Mammalian target of rapamycin complex 1 (mTORC1) is implicated in cell growth control and is extensively regulated. We previously reported that in response to hypoxia, mTORC1 is inhibited by the protein regulated in development and DNA damage response 1 (REDD1). REDD1 is upregulated by HIF-1, and forced REDD1 expression is sufficient to inhibit mTORC1. REDD1-induced mTORC1 inhibition is dependent on a protein complex formed by the tuberous sclerosis complex (TSC)1 and 2 (TSC2) proteins. In clear-cell renal cell carcinoma (ccRCC), the von Hippel-Lindau (VHL) gene is frequently inactivated leading to constitutive activation of HIF-2 and/or HIF-1, which may be expected to upregulate REDD1 and inhibit mTORC1. However, mTORC1 is frequently activated in ccRCC and mTORC1 inhibitors are effective against this tumor type; a paradox herein examined. REDD1 was upregulated in VHL-deficient ccRCC by in silico microarray analyses, as well as by quantitative real-time PCR, Western blot, and immunohistochemistry. Vhl disruption in a mouse model was sufficient to induce Redd1. Using ccRCC-derived cell lines, we show that REDD1 upregulation in tumors is VHL-dependent, and that both HIF-1 and HIF-2 are, in a cell-type dependent manner, recruited to, and essential for, REDD1 induction. Interestingly, whereas mTORC1 is responsive to REDD1 in some tumors, strategies have evolved in others, such as mutations disrupting TSC1, to subvert mTORC1 inhibition by REDD1. Sequencing analyses of 77 ccRCCs for mutations in TSC1, TSC2 and REDD1, using PTEN as a reference, implicate the TSC1 gene, and possibly REDD1, as tumor suppressors in sporadic ccRCC. Understanding how ccRCCs become refractory to REDD1-induced mTORC1 inhibition should shed light into the development of ccRCC and may aid in patient selection for molecular targeted therapies.
PMCID: PMC3234675  PMID: 21798997
REDD1; DDIT4; mTORC1; VHL; RCC; HIF; nucleolus
12.  Cost-Effectiveness Analysis of Stereotactic Body Radiation Therapy Versus Intensity-Modulated Radiation Therapy: An Emerging Initial Radiation Treatment Option for Organ-Confined Prostate Cancer 
Journal of Oncology Practice  2012;8(3 Suppl):e31s-e37s.
Stereotactic body radiation therapy for low- to intermediate-risk prostate cancer has potential cost savings and may improve access to radiation, increase convenience, and boost quality of life.
The purpose of this study is to compare the cost-effectiveness of two external beam radiation therapy techniques for treatment of low- to intermediate-risk prostate cancer: stereotactic body radiation therapy (SBRT) and intensity-modulated radiation therapy (IMRT).
Materials and Methods:
A Markov decision analysis model with probabilistic sensitivity analysis was designed with the various disease states of a 70-year-old patient with organ-confined prostate cancer to evaluate the cost-effectiveness of two external beam radiation treatment options.
The Monte Carlo simulation revealed that the mean cost and quality-adjusted life-years (QALYs) for SBRT and IMRT were $22,152 and 7.9 years and $35,431 and 7.9 years, respectively. The sensitivity analysis revealed that if the SBRT cohort experienced a decrease in quality of life of 4% or a decrease in efficacy of 6%, then SBRT would no longer dominate IMRT in cost-effectiveness. In fact, with these relaxed assumptions for SBRT, the incremental cost-effectiveness ratio of IMRT met the societal willingness to pay threshold of $50,000 per QALY.
Compared with IMRT, SBRT for low- to intermediate-risk prostate cancer has great potential cost savings for our health care system payers and may improve access to radiation, increase patient convenience, and boost quality of life for patients. Our model suggests that the incremental cost-effectiveness ratio of IMRT compared with SBRT is highly sensitive to quality-of-life outcomes, which should be adequately and comparably measured in current and future prostate SBRT studies.
PMCID: PMC3348607  PMID: 22942832
13.  Statistical consideration for clinical biomarker research in bladder cancer 
Urologic oncology  2010;28(4):389-400.
To critically review and illustrate current methodologic and statistical considerations for bladder cancer biomarker discovery and evaluation.
Original, review, and methodological articles, and editorials were reviewed and summarized.
Biomarkers may be useful at multiple stages of bladder cancer management: early detection, diagnosis, staging, prognosis, and treatment; however, few novel biomarkers are currently used in clinical practice. The reasons for this disjunction are manifold and reflect the long and difficult pathway from candidate biomarker discovery to clinical assay, and the lack of coherent and comprehensive processes (pipelines) for biomarker development. Conceptually, the development of new biomarkers should be a process that is similar to therapeutic drug evaluation - a highly regulated process with carefully regulated phases from discovery to human applications. In a further effort to address the pervasive problem of inadequacies in the design, analysis, and reporting of biomarker prognostic studies, a set of reporting recommendations are discussed. For example, biomarkers should provide unique information that adds to known clinical and pathologic information. Conventional multivariable analyses are not sufficient to demonstrate improved prediction of outcomes. Predictive models, including or excluding any new putative biomarker, needs to show clinically significant improvement of performance in order to claim any real benefit. Towards this end, proper model building, avoidance of overfitting, and external validation are crucial. In addition, it is important to choose appropriate performance measures dependent on outcome and prediction type and to avoid use of cut-points. Biomarkers providing a continuous score provide potentially more useful information than cut-points since risk fits a continuum model. Combination of complementary and independent biomarkers is likely to better capture the biologic potential of a tumor than any single biomarker. Finally, methods that incorporate clinical consequences such as decision curve analysis are crucial to the evaluation of biomarkers.
Attention to sound design and statistical practice should be delivered as early as possible and will help maximize the promise of biomarkers for patient care. Studies should include a measure of predictive accuracy and clinical decision-analysis. External validation using data from an independent cohort provides the strongest evidence that a model is valid. There is a need for adequately assessed clinical biomarkers in bladder cancer.
PMCID: PMC3407571  PMID: 20610277
biomarker; diagnosis; prognosis; treatment; nomogram; decision-analysis; bladder cancer; statistics; statistical analysis
14.  Detection of bladder cancer using novel DNA methylation biomarkers in urine sediments 
Bladder cancer remains a lethal malignancy that can be cured if detected early. DNA hypermethylation is a common epigenetic abnormality in cancer that may serve as a marker of disease activity.
We selected 10 novel candidate genes from the most frequently hypermethylated genes detected by DNA microarray and bisulfite pyrosequencing of bladder cancers and applied them to detect bladder cancer in urine sediments. We analyzed DNA methylation in the candidate genes by quantitative methylation specific real time PCR (qMSP) to detect bladder cancer in urine sediments from 128 bladder cancer patients and 110 age-matched control subjects.
Based on a multi-gene predictive model, we discovered 6 methylation markers (MYO3A, CA10, SOX11, NKX6-2, PENK and DBC1) as most promising for detecting bladder cancer. A panel of 4 genes (MYO3A, CA10, NKX6-2 and DBC1 or SOX11) had 81 % sensitivity and 97 % specificity, while a panel of 5 genes (MYO3A, CA10, NKX6-2, DBC1 and SOX11 or PENK) had 85 % sensitivity and 95 % specificity for detection of bladder cancer (AUC=0.939). Analyzing the data by cancer invasiveness, detection rate was 47 out of 58 (81 %) in non-muscle invasive tumors (pTa, Tis and pT1) and 62 out of 70 (90 %) in muscle invasive tumors (T2, T3 and T4).
This biomarker panel analyzed by qMSP may help the early detection of bladder tumors in urine sediments with high accuracy.
The panel of biomarker deserves validation in a large well-controlled prospectively collected sample set.
PMCID: PMC3132294  PMID: 21586619
15.  Practice Variation in the Surgical Management of Urinary Lithiasis 
The Journal of Urology  2011;186(1):146-150.
SWL and URS are highly effective treatments for urinary lithiasis. While stone size and location are primary determinants of therapy, little is known about other factors associated with treatment. Our objective was to identify patient, provider and practice setting characteristics associated with selection of ureteroscopy or SWL.
We used the Medicare 5% sample to identify beneficiaries with an incident stone encounter from 1997 –2007. Within this group, we identified beneficiaries undergoing SWL or URS for the management of urinary calculi. Multivariable regression models identified factors associated with use of URS.
The cohort comprised 9358 beneficiaries who underwent an initial procedure. SWL was used in 5208 (56%) beneficiaries, while URS was used in 4150 (44%). Females were less likely than males to undergo URS (OR 0.844, p = 0.006). Providers who more recently completed residency training employed URS more often (p = 0.023). Provider and facility volume were associated with initial procedure selection. The odds of a second procedure following initial SWL were 1.54 times that of URS (p<0.001).
Non-clinical factors are associated with use of URS or SWL for initial stone management, which may reflect provider and/or patient preferences or experience. Further investigation is required to understand the impact of these outcomes on quality and cost of care.
PMCID: PMC3280611  PMID: 21575964
ureteroscopy; shock wave lithotripsy; outcomes
16.  Phase I Dose-Escalation Study of Stereotactic Body Radiation Therapy for Low- and Intermediate-Risk Prostate Cancer 
Journal of Clinical Oncology  2011;29(15):2020-2026.
To evaluate the tolerability of escalating doses of stereotactic body radiation therapy in the treatment of localized prostate cancer.
Patients and Methods
Eligible patients included those with Gleason score 2 to 6 with prostate-specific antigen (PSA) ≤ 20, Gleason score 7 with PSA ≤ 15, ≤ T2b, prostate size ≤ 60 cm3, and American Urological Association (AUA) score ≤ 15. Pretreatment preparation required an enema and placement of a rectal balloon. Dose-limiting toxicity (DLT) was defined as grade 3 or worse GI/genitourinary (GU) toxicity by Common Terminology Criteria of Adverse Events (version 3). Patients completed quality-of-life questionnaires at defined intervals.
Groups of 15 patients received 45 Gy, 47.5 Gy, and 50 Gy in five fractions (45 total patients). The median follow-up is 30 months (range, 3 to 36 months), 18 months (range, 0 to 30 months), and 12 months (range, 3 to 18 months) for the 45 Gy, 47.5 Gy, and 50 Gy groups, respectively. For all patients, GI grade ≥ 2 and grade ≥ 3 toxicity occurred in 18% and 2%, respectively, and GU grade ≥ 2 and grade ≥ 3 toxicity occurred in 31% and 4%, respectively. Mean AUA scores increased significantly from baseline in the 47.5-Gy dose level (P = .002) as compared with the other dose levels, where mean values returned to baseline. Rectal quality-of-life scores (Expanded Prostate Cancer Index Composite) fell from baseline up to 12 months but trended back at 18 months. In all patients, PSA control is 100% by the nadir + 2 ng/mL failure definition.
Dose escalation to 50 Gy has been completed without DLT. A multicenter phase II trial is underway treating patients to 50 Gy in five fractions to further evaluate this experimental therapy.
PMCID: PMC3138546  PMID: 21464418
17.  High Risk Patients with Hematuria Are Not Evaluated According to Guideline Recommendations 
Cancer  2010;116(12):2954-2959.
To determine whether high risk patients with hematuria receive evaluation according to guideline recommendations.
Materials and Methods
We recently performed a screening study for bladder cancer using a urine-based tumor marker in 1502 subjects at high risk based on age over 50, ≥10-year smoking history, and/or a 15 year or more environmental exposure. We evaluated use of urinalysis (UA) within 3 years preceding the screening study. Chart review was performed to determine if this subset with microhematuria received any additional evaluation.
Of 1502 study participants, routine urinalysis was performed in 73.2% and 164 (14.9%) subjects had documented hematuria (>3 RBCs/HPF) prior to inclusion. Of these, 42.1% had no further evaluation. Additional testing included repeat urinalysis (36%), urine culture (15.2%), cytology (10.4%), imaging (22.6% overall: 15.9% CT, 4.3% IVP; 2.4% MRI) and cystoscopy (12.8%).
Three subjects with microscopic hematuria (2%) were subsequently found to have bladder cancer during the screening study but were not referred for evaluation based on their hematuria. The source of hematuria was unknown in 65%, infection in 22%, benign prostatic enlargement in 10% and renal stone disease in 4% but these results are based on incomplete evaluation since only 12.8% underwent cystoscopy.
Subjects at high risk for bladder cancer based on ≥10 years of smoking or environmental exposure with microscopic hematuria are rarely evaluated thoroughly and only 12.8% were referred for urologic evaluation. Further studies are needed to evaluate both the utilization and effectiveness of guidelines for hematuria.
PMCID: PMC2940122  PMID: 20564400
Hematuria; Guidelines Recommendations; bladder cancer
18.  Lymphovascular Invasion Predicts Clinical Outcomes in Patients With Node-Negative Upper Tract Urothelial Carcinoma 
Journal of Clinical Oncology  2008;27(4):612-618.
To assess the association of lymphovascular invasion (LVI) with cancer recurrence and survival in a large international series of patients treated with radical nephroureterectomy (RNU) for upper urinary tract urothelial carcinoma (UTUC).
Patients and Methods
Data were collected on 1,453 patients treated with RNU at 13 academic centers and combined into a relational database. Pathologic slides were rereviewed by genitourinary pathologists according to strict criteria. LVI was defined as presence of tumor cells within an endothelium-lined space.
LVI was observed in 349 patients (24%). Proportion of LVI increased with advancing tumor stage, high tumor grade, presence of tumor necrosis, sessile tumor architecture, and presence of lymph node metastasis (all P < .001). LVI was an independent predictor of disease recurrence and survival (P < .001 for both). Addition of LVI to the base model (comprising pathologic stage, grade, and lymph node status) marginally improved its predictive accuracy for both disease recurrence and survival (1.1%, P = .03; and 1.7%, P < .001, respectively). In patients with negative lymph nodes and those in whom a lymphadenectomy was not performed (n = 1,313), addition of LVI to the base model improved the predictive accuracy of the base model for both disease recurrence and survival by 3% (P < .001 for both). In contrast, LVI was not associated with disease recurrence or survival in node-positive patients (n = 140).
LVI was an independent predictor of clinical outcomes in nonmetastatic patients who underwent RNU for UTUC. Assessment of LVI may help identify patients who could benefit from multimodal therapy after RNU. After confirmation, LVI should be included in staging of UTUC.
PMCID: PMC2737380  PMID: 19075275
19.  Persistent Uroplakin Expression in Advanced Urothelial Carcinomas: Implications in Urothelial Tumor Progression and Clinical Outcome 
Human pathology  2007;38(11):1703-1713.
As the terminal differentiation products of human urothelium, uroplakins (UPs) would be expected to diminish during urothelial tumorigenesis. Surprisingly, recent studies found UPs to be retained even by well-advanced urothelial carcinomas, suggesting that the loss of UPs does not strictly parallel urothelial transformation. Little is known, however, about whether the status of UPs is associated with a particular pathological parameter, tumor’s biological behavior or patient outcome. Here we assessed UP expression by immunohistochemistry on tissue arrays from 285 patients with bladder urothelial carcinomas or non-tumor conditions. UPs were expressed in all 9 normal urothelial specimens, 63/74 (85%) patients with non-muscle-invasive urothelial carcinomas on transurethral resection, 104/202 (51.5%) patients who underwent radical cystectomy for advanced urothelial carcinomas, and 33/50 (66%) lymph node metastases. Normally associated with urothelial apical surface, UPs were localized aberrantly in tumors, including micro-luminal, basal-laminal, cytoplasmic or uniform patterns. In non-muscle-invasive diseases, there was no association between UP expression and disease recurrence, progression or mortality. In contrast, in invasive diseases, absent UP expression was significantly associated with advanced pathologic stage, lymph node metastases, disease recurrence and bladder cancer-specific mortality (p=0.042, p=0.035, p=0.023 and p=0.022, respectively) in univariate analyses. Furthermore, UP status was independent of key cell-cycle regulators, including p53, pRb, p27 and cyclin D1, thus excluding a functional link between these two groups of proteins. Our data demonstrate for the first time that persistent UP expression is associated with a favorable clinical outcome and that UPs may be used as adjunct markers for predicting the prognoses of patients with invasive and metastatic bladder carcinomas. Our results also suggest that UP-positive and –negative carcinomas have different clonal origins or may be derived from different cancer stem cells.
PMCID: PMC2778836  PMID: 17707461
bladder cancer; uroplakin; progression; prognosis; transitional cell carcinoma
20.  Quantitation of Aurora Kinase A Gene Copy Number in Urine Sediments and Bladder Cancer Detection 
Chromosome missegregation and the resulting aneuploidy is a common change in neoplasia. The Aurora kinase A (AURKA) gene, which encodes a key regulator of mitosis, is frequently amplified and/or overexpressed in cancer cells, and the level of AURKA amplification is associated with the level of aneuploidy. We examined whether AURKA gene amplification is a biomarker for the detection of bladder cancer.
The effect of ectopic expression of Aurora kinase A (AURKA) using an adenoviral vector in simian virus 40–immortalized urothelial cells (SV-HUC) on centrosome multiplication and chromosome copy number was measured in vitro by immunofluorescence and fluorescence in situ hybridization (FISH), respectively. The FISH test was also used to examine AURKA gene copy number in exfoliated cells in voided urine samples from 23 patients with bladder cancer and 7 healthy control subjects (training set), generating a model for bladder cancer detection that was subsequently validated in an independent set of voided urine samples from 100 bladder cancer patients and 148 control subjects (92 healthy individuals and 56 patients with benign urologic disorders). An AURKA gene score (the proportion of cells with three or more AURKA signals) was used to produce receiver operating characteristic (ROC) curves and to calculate the specificity and sensitivity of the AURKA FISH test. Differences between mean AURKA scores in different pathogenetic groups of bladder cancer stratified according to histological grade and stage were tested by unpaired Mann–Whitney t tests or one-way Wilcoxon tests. All statistical tests were two-sided.
Forced overexpression of AURKA in urothelial cells induced amplification of centrosomes, chromosome missegregation, and aneuploidy, and natural overexpression was detectable in in situ lesions from patients with bladder cancer. The FISH test for the AURKA gene copy number performed on the validation set yielded a specificity of 96.6% (95% confidence interval [CI] = 92.3% to 98.5%) and sensitivity of 87% (95% CI = 79.0% to 92.2%) and an area under the ROC curve of 0.939 (95% CI = 0.906 to 0.971; P < .001).
Overexpression of AURKA can cause aneuploidy in urothelial cells, and the AURKA gene copy number is a promising biomarker for detection of bladder cancer.
PMCID: PMC2720731  PMID: 18812553
22.  Laparoscopic Partial Nephrectomy Using Holmium Laser in a Porcine Model 
To evaluate the utility of the holmium laser for partial nephrectomy in a porcine model.
Transperitoneal lower pole laparoscopic partial nephrectomy was performed in 5 farm pigs. All animals underwent a left-sided laparoscopic partial nephrectomy and were kept alive for 2 weeks (survival group). Subsequently, a right laparoscopic partial nephrectomy was performed (acute group), and the animals were sacrificed. A 1000-μm (n=6) or 550-μm (n=4) end-fire holmium laser fiber set at 0.2 joules and 60 pulses per second was used to transect the lower pole of the kidney 1 cm below the level of the hilum. The cut parenchymal surface was then sealed with fibrin glue in the survival animals. The operated on kidneys were inspected grossly and evaluated microscopically.
Laser transection was successfully completed in all cases, and hemostasis proved adequate without any adjunctive measures. No perioperative complications occurred. Estimated blood loss was less than 50 cc for each laparoscopic partial nephrectomy. The acute and survival pigs showed no statistically significant differences in specimen size or weight. Serum creatinine levels were normal in all survival animals. Extravasation was noted on retrograde pyelograms of 2 animals in the survival group.
The Holmium:YAG laser provides an efficacious modality for transecting the kidney in a porcine model. Clinical trials are necessary to determine its role in laparoscopic partial nephrectomy in humans.
PMCID: PMC3015512  PMID: 14974664
Holmium laser; Partial nephrectomy; Laparoscopy; Kidney; Porcine model
23.  Laparoscopic Treatment of Ovarian Vein Syndrome 
Ovarian vein syndrome is a rare cause of ureteral obstruction. In this report, we describe an unusual presentation of the syndrome successfully treated with laparoscopic techniques.
The patient presented with a 12-month history of right flank pain and a right abdominal mass. The preoperative evaluation revealed renal malrotation, hydronephrosis, decreased renal function, and presumed ureteropelvic junction obstruction.
By using a transperitoneal laparoscopic approach, an enlarged ovarian vein was identified as the cause of the ureteral obstruction. The ovarian vein was divided with a laparoscopic stapler. The patient's postoperative course was unremarkable, and she was discharged from the hospital on the second postoperative day. At 3-months follow-up, the patient was completely asymptomatic without evidence of obstruction.
Ovarian vein syndrome remains a rare diagnosis of exclusion. A careful preoperative evaluation is required to exclude other causes of ureteral obstruction. By using a laparoscopic approach, the ureter and obstructing vessel were readily identified to effectively treat the patient. With the minimally invasive approach, postoperative recovery and patient quality of life were improved.
PMCID: PMC3113207  PMID: 14558716
Laparoscopy; Ureteral obstruction; Ovarian vein syndrome
24.  Laparoscopic Nephrectomy Is Cost Effective Compared With Open Nephrectomy in a Large County Hospital 
Background and Objectives:
To evaluate the experience with laparoscopic nephrectomy in a large county hospital and perform a cost comparison between uncomplicated open and laparoscopic nephrectomy.
Eleven consecutive patients who underwent an uncomplicated laparoscopic nephrectomy in a large county hospital were compared with 8 patients who underwent uncomplicated open nephrectomy during the same period. Patient charts and corresponding billing records were reviewed to determine overall hospitalization cost and individual cost components.
No perioperative complications occurred in either the laparoscopic or open group, and no statistically significant differences existed between groups with regard to patient demographics or operative parameters. The overall operating room costs favored the open nephrectomy group by $1070 (P=0.003). However, the overall cost of hospitalization, surgeon professional fees, duration of hospitalization, room and board costs, laboratory, and radiology costs, pharmacy costs, intravenous solution and infusion pump costs all significantly favored the laparoscopic patient group. The mean difference in overall hospital cost between laparoscopic and open nephrectomy was $1211 in favor of laparoscopy (P=0.037).
Our experience with laparoscopic nephrectomy in a large county hospital demonstrates a clear economic advantage in favor of the laparoscopic approach. Given limited funding for public hospitals and a clear patient benefit, laparoscopic nephrectomy should constitute first-line therapy when nephrectomy is indicated.
PMCID: PMC3015483  PMID: 12856840
Laparoscopic nephrectomy; Cost comparison

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